PROJECT SUMMARY/ABSTRACT Pathogenic microorganisms are the cause of diverse infections and diseases worldwide. The emergence of drug resistant strains increases the infection and mortality rates and demands new approaches and efforts in the antimicrobial drug discovery. The long-term goal of our research is to understand the role of molecular chaperones in pathogen growth, survival, and virulence and to apply that knowledge in developing molecular approaches to combat infectious diseases. The objective of this application is to discover small-molecule inhibitors of Hsp100 chaperones using high-throughput library screening. The Hsp100 chaperones reactivate aggregated cellular proteins. A loss of Hsp100 is detrimental for infectivity and survival of a number of bacterial and protozoan pathogens. Importantly, no apparent Hsp100 orthologs are found in metazoan proteomes. As the primary target for inhibitor development and testing, we will use the Hsp100 chaperone, ClpB, from Shigella/Escherichia coli. The following Specific Aims will be pursued: 1. We will perform a sequence of two screens of a small-molecule library that will interrogate two orthogonal functionalities of ClpB: ATP-dependent substrate binding and substrate-induced activation of the ATPase activity. 2. We will validate and prioritize compound hit chemotypes that inhibit ClpB using medicinal chemistry approaches. 3. We will validate the inhibitory potency of selected hit compounds and test their selectivity towards ClpB in vitro by performing a number of orthogonal biochemical assays. 4. We will perform preliminary testing of the antimicrobial activity of the hit compounds in Gram-negative bacteria Escherichia coli, Shigella flexneri, and Shigella sonnei, and Gram-positive Staphylococcus aureus. The expected outcome of the proposed studies will be the discovery of validated small-molecule Hsp100-inhibitor candidates for future medicinal chemistry optimization and biological testing. This approach is innovative because no chaperone-based antimicrobials have been implemented yet.